1. Field of the Invention
The present invention relates to the precursor protein of amyloid plaque core (APC) polypeptide, to fragments of the precursor protein and to the diagnostic use of the precursor protein and of the fragments. Furthermore, the invention relates to the DNA coding for the precursor protein, to fragments of this DNA and to the diagnostic use of the DNA and of the fragments.
Alzheimer's disease was described as an independent clinical and pathological entity for the first time in the year 1907 by the German neurologist Alois Alzheimer (Alzheimer, A. (1907) Zentralblatt fur Nervenheilkunde und Psychiatrie, 177-179). It is the commonest degenerative brain disease of old people. In America alone about 2 million people are now suffering from the disease, and at least 100,000 die of it each year (Wurtman, R. J. (1985) Sci. Am. 252, 48-56).
The disease appears in people between 40 and 80 years of age. Those affected gradually lose their memory and their ability to concentrate. The state of mental deterioration advances until, within 3 to 10 years, the patients are unable either to speak, to think or to take care of themselves, and finally they die. The cause of this dementia is unknown. There is neither a definitive diagnosis nor a therapy.
Brain autopsies of people who have died of Alzheimer's disease reveal typical changes under the microscope as follows:
There has been a decrease in the number of neurons, especially in the parietal lobes, that is to say in the parts of the brain where the memory functions are localized. A loss of neurons which normally release acetylcholine is likewise clearly visible.
In addition, three extremely unusual structures appear in the cerebral cortex, these structures not existing in the brain of healthy people and thus being used for diagnosis (after death):
1) intracellular neurofibrils
(NFTs, neurofibrillary tangles)
In the cytosome of neurons of the cerebral cortex and of the hippocampus are found bundles consisting of two filaments which are twisted around one another in the manner of a helix (PHFs, paired helical filaments).
2) extracellular amyloid plaques
(APC, amyloid plaque core)
The neuritic plaques contain amyloid and the residues of dead cells, and they are scattered over the cerebral cortex, the hippocampus and the amygdaloid nucleus. The number of plaques is correlated with the degree of mental deterioration.
3) cerebrovascular amyloid
(ACA, amyloid congophilic angiopathy)
Amyloid is the name given to a protein-rich composition. Such amorphous protein aggregates are to be found all around the blood vessels and in the wall of blood vessels in the brain.
The protein component of ACA has been isolated and sequenced (Glenner, G. G. & Wong, C. W. (1984) Biochem. Biophys. Res. Commun. 120, 885-890). The amino acid sequence has no homology with known protein sequences. The protein components of PHFs and APC have likewise been isolated and sequenced (Masters, C. L., Multhaupt, G., Simms, G., Pottgiesser, J., Martins R. N. and Beyreuther, K. (1985) EMBO 4, 2757-2763 and Masters, C. L., Simms, G., Weinman, N. A., Multhaupt, G., McDonald, B. L. and Beyreuther, K. (1985) Proc. Natl. Acad. Sci. USA 82, 4245-4249). The amino acid sequences indicate that all three polypeptides are probably the same one having a molecular weight of 4.5 kD. The relevant sequence is shown in boxes in FIGS. 1a-c (positions 597-638).
There are several hypotheses to explain the origin of this APC protein. It might be a normal protein in the brain (or even in another organ) in which either regulation of biosynthesis has become deranged or physiological breakdown is impaired. The accumulations of very large amounts might then be the cause of the disease. If it is an abnormal protein, and its unusual ability to aggregate causes the disease, it might also be coded for by a healthy human gene which was under faulty control due to some factor or other, for example, viruses, foodstuffs or environmental toxins. The fault might also comprise a modification of the original protein precursor. On the other hand however, a viral gene might also be responsible for synthesizing the APC protein.
In the work leading to the invention an attempt has now been made to establish the origin and nature of the APC protein, whose aggregation in the cerebral cortex is one of the main biochemical signs in Alzheimer patients, in order thereby to obtain a tool for improved diagnosis of Alzheimer's disease.
For this purpose, a human fetal brain c-DNA bank with pA+mRNA of the cerebral cortex was constructed.
The c-DNA was synthesized by the method of Okayama and Berg (Okayama, H. and Berg, P. Mol. Cell. Biol. 2, 161-170 (1982); Okayama, H. and Berg, P. Mol. Cell. Biol. 3, 280-289 (1983)), and the c-DNA was transformed into E. coli HB 101 (Aviv, H. and Leder, P. Proc. Natl. Acad. Sci. USA 69, 1408 (1972)). Each of the c-DNA banks obtained in this way contains more than 1.times.10.sup.6 independent c-DNA clones.
To screen the bank, use was made of a DNA probe whose sequence was derived from the sequence of APC polypeptide. The chosen sequence corresponds to the amino acids in positions 10-16 of APC. The relevant sequence is indicated by a brace in FIG. 1c (positions 1815-1835). In order to ensure optimum hybridization, the degeneracy of the genetic code was taken into account, and a mixture having the following sequence ##STR1## was prepared and used as probe. This is a 64-fold degenerate 20-mer. A test on 100,000 c-DNA clones from the human fetal cerebral cortex bank resulted in the isolation of a complete (full-length) c-DNA clone, having the serial No. EC 9.110, which codes for a protein which contains the APC sequence and thus represents the precursor protein of APC peptide. The sequence of the c-DNA, and the amino acid sequence of the coded protein, are to be found in FIG. 1. Sequence analysis was carried out by the dideoxy method (Sanger, F., Nicklen, S. and Coulson, A. R. Proc. Natl. Acad. Sci. USA 74, 5463-5467 (1977) and Guidelines for quick and simple Plasmid Sequencing, Handbook, (1986) Boehringer Mannheim GmbH, Biochemica, D-6800 Mannheim). Nothing is known at present about the natural function of the APC precursor protein.